Removal of antibiotic resistant genes in wastewater treatment plants

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FACULTY OF SCIENCE AND TECHNOLOGY

MASTER’S THESIS

Study programme/specialisation:

Environmental Engineering Spring semester, 2020

Open Author: Rebekka Bryne Bolme

….………..…

(signature of author) Programme

coordinator: Roald Kommedal

Supervisor: Krista Michelle Kaster

Title of master’s thesis: Removal of antibiotic resistant genes in wastewater treatment plants

Credits: 30

Keywords: Wastewater treatment, antibiotic resistant genes, HGT, ARG removal, PCR, Constructed Wetlands, MBR, AOPs

Number of pages: 77 + supplemental material/other: 12

Stavanger, 29/06/2020 date/year

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REMOVAL OF ANTIBIOTIC RESISTANT GENES IN WASTEWATER TREATMENT PLANTS

MASTER’S THESIS

WATER SCIENCE AND TECHNOLOGY

ENVIRONMENTAL TECHNOLOGY STUDY PROGRAM DEPARTMENT OF MATHEMATICS AND NATURAL SCIENCES

UNIVERSITY OF STAVANGER

2020

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ABSTRACT

Antibiotic resistance has been regarded as a growing international problem, due to extensive overuse of antibiotics has led to untreatable infections caused by antibiotic-resistant bacteria (ARB). Wastewater treatment plants (WWTPs) have gained focus as critical hot spots for resistance due to the suited environment for the proliferation of antibiotic-resistant genes (ARGs). Promising removal values have been detected during advanced wastewater treatment by membrane bioreactors (MBRs). Further knowledge into the mechanism of removal and proliferation of ARGs and mobile genetic elements (MGEs) is needed to determine an optimal treatment method for remediation of antibiotic resistance. In this research, the study of the occurrence of antibiotic resistance during several stages of Mekjavik wastewater treatment plant in the Rogaland county in Norway was performed. Resistance to two types of antibiotics has been evaluated by DNA extraction from wastewater inlet, outlet and activated sludge followed by genomic analysis using polymerase chain reaction (PCR). Sulphonamides and Tetracyclines the resistance encoding genes Sul 1, 2, 3, as well as Tet A, B, C, D, E, G, L, K, M, O, were qualitatively analysed. The genes coding for Tetracycline tet B tet O were detected in the inlet, outlet, and sludge samples. The gene tet C was not detected in any stages of the wastewater treatment plant.

Keywords: Wastewater treatment, antibiotic resistant genes, HGT, ARG removal, PCR, Constructed Wetlands, MBR, AOPs

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ACKNOWLEDGEMENTS

I would love to thank my family and friends for the constant support during the work with my master’s thesis. I also want to express my gratitude to my advisor, Associate Professor Krista Michelle Kaster, for her helpful advice and guidance during this time. Lastly, I want to thank all my fellow students who have made the time at the University of Stavanger so enjoyable.

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LIST OF FIGURES

Figure 1- Classes of antibiotics used in Europe specific to human medicine. ... 4

Figure 2 - Antibiotic targets and bacterial mechanisms of resistance. ... 9

Figure 3 - Mechanisms of vertical and lateral gene transfer in bacteria. ... 10

Figure 4 – Route of antibiotics from human and animal consumption through WWTPs to the environment and the resulting resistance problem in clinical settings. ... 15

Figure 5 – Etest antibiotic susceptibility test. ... 20

Figure 6 - Fate of antibiotic resistance in Conventional AS treatment processes. ... 26

Figure 7 - Removal mechanisms of pollutants in constructed wetlands ... 30

Figure 8 - Membrane fouling in anaerobic MBR. ... 35

Figure 9 – Gel electrophoresis illustration PCR fragments tetB and tetC ... 44

Figure 10- Gel electrophoresis illustration PCR fragments tetO ... 44

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LIST OF TABLES

Table 2-1 Antibiotics and their target area. ... 6

Table 2-2 MBR antibiotic resistance determinant removal efficiencies. ... 34

Table 2-3 Hydroxyl radical producing reactions in advanced oxidation processes ... 37

Table 2-4 AOPs removal of ARGs and operational parameters ... 38

Table 3-1 Sulphonamide resistant genes ... 41

Table 3-2 Tetracycline resistant genes primers and PCR conditions ... 42

Table 4-1 Antibiotic resistant genes verified in SNJ wastewater ... 43

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ABBREVIATIONS

WHO Word Health Organization ARG Antibiotic Resistant Genes ARB Antibiotic Resistant Bacteria WWTP Wastewater Treatment Plant OMP Organic Micropollutants HGT Horizontal gene transfer MGE Mobile Genetic elements DNA Deoxy Ribonucleic Acid PCR Polymerase Chain reaction MDR Multidrug Resistance RNA Ribonucleic Acid

EPPP Environmental Persistent Pharmaceutical Pollutants MIC Minimum Inhibitory Concentration

qPCR Quantitative Polymerase Chain Reaction PMA Propidium Monoazide

CAS Conventional Activated Sludge AD Anaerobic digestion

CW Constructed Wetland

FSF- CW Free Surface-Flow Constructed Wetland

VSSF-CW Vertical Subsurface-Flow Constructed Wetland HSSF-CW Horizontal Subsurface-Flow Constructed Wetland ICW Integrated Constructed Wetland

VUF- CW Vertical Upflow Constructed Wetland

UV Ultraviolet

Cl Chlorination

AOP Advanced Oxidation Process MBR Membrane Bioreactor

UF Ultrafiltration MF Microfiltration

TMP Transmembrane Pressure LRV Log Reduction Value

SNJ Sentralrenseanlegg Nord-Jæren

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1.0 INTRODUCTION

Antibiotic resistance has been regarded as a threat to global health (World Health Organization, 2014). Thousands of deaths each year in the EU alone have been attributed to pathogenic ARB, making infections untreatable (Rizzo et al., 2013). WWTPs have become a focus of concern because of the massive amounts of microorganisms and antibiotics coming in through the pipelines and the favourable environment for horizontal gene transfer (HGT) of ARGs and proliferation in the plants (Rizzo et al., 2013). WWTPs have been regarded as point contamination sources, contributing to the selective pressure in the surrounding environmental compartments upon release of effluents containing large amounts of ARB and ARGs (Pazda, Kumirska, Stepnowski, & Mulkiewicz, 2019). ARGs have been found in various environmental matrices (Sharma, Johnson, Cizmas, McDonald, & Kim, 2016). The removal of ARGs during the stages of wastewater treatment has shown to be critical for the health of the organisms surrounding the sources of outlets of the plants (Xue et al., 2019).

International urgency for surveillance and monitoring of antimicrobial resistance has emerged, in addition to the need to optimise the methods for detection, quantification and proliferation of antibiotic resistance determinants (Berendonk et al., 2015). Comparability and standardisation of detecting ARBs and ARGs in environmental samples, including wastewater treatment plants, have not yet been fully established (Rizzo et al., 2013). The need for further investigation to develop repeatable and reliable analysing tools to build databases on antibiotic resistance occurrence, as well as practical and efficient treatment methods for removal of ARGs have in recent years been regarded as a matter of environmental and public health (Sarode et al., 2019).

Tetracyclines and Sulphonamides have been some of the most widely used classes of antibiotics in the world, and the occurrence of ARGs in bacteria with resistance to these compounds have been frequently detected in WWTPs (Xue et al., 2019). Sulphonamide resistant genes (sul1 and sul2) have been suggested as monitoring targets by the European Cooperation in Science and Technology (Berendonk et al., 2015). These factors were the motivation for the choice of ARGs encoding for tetracyclines and sulphonamides as targets for qualitative analysis in different wastewater treatment matrices during this study.

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1.1 Objectives

The objective of this review was to give a review of current conventional wastewater treatment technologies efficiencies in ARG removal and to evaluate the possibility of improvement by advanced treatment processes.

A selection of ARGs coding for resistance to Sulphonamide and Tetracycline was qualitatively detected in a conventional biological treatment plant to evaluate the applicability of molecular- based PCR method for ARG detection and the occurrence of these genes during different stages of the wastewater treatment plant.

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2.0 LITERATURE REVIEW

2.1 Antibiotics

Antibiotics are naturally occurring, semi-synthetic or synthetically made chemicals used to inhibit growth or cause death in microorganisms (Homem & Santos, 2011). Ever since the discovery of the first antibiotic in the early 1900s, the medical field has possessed a precious weapon in treating bacterial infections (Pruden et al., 2013). The use of antibiotics have increased as the global healthcare system has improved during the last century (Al-Riyami, Ahmed, Al-Busaidi, & Choudri, 2018). There has been an increasing trend of misuse of antimicrobial agents in human medicine, due to lack of education regarding antibiotic use and regulation of prescription drugs and over-the-counter sales (Lood, Ertürk, & Mattiasson, 2017).

Systematic data collection concerning the distribution of clinical and personal use of antimicrobial agents on a global basis have been scarce, especially in developing countries (Sosa, 2010, p. 7). A study of global human consumption in the year 2010 showed over 70 billion standard units in the period 2000 to 2010, which was a 35 % increase from the previous decade (Van Boeckel et al., 2014).

Antibiotic use in recent years in animals was found to be double that of human consumption (Van Boeckel et al., 2015). Advances in the optimisation of agricultural livestock industry have been partly attributed to the extensive use of antibiotics to prevent diseases and for growth promotion purposes (Graham, Boland, & Silbergeld, 2007). Overuse of these agents has led to the cumulative release of antibiotics into surrounding environments (Xu et al., 2007), challenging environmental microbial populations (Kurt, Mert, Özengin, Sivrioğlu, & Yonar, 2017). Additively, based on bio-accumulation in the environment due to their non- biodegradability, antibiotics have been categorised as emerging pollutants (Martinez, 2009).

The classes of antibiotics that are predominantly utilised in clinical and agricultural purposes today are β-lactams (including penicillins and cephalosporins), macrolides, quinolones, sulphonamides, trimethoprim and tetracyclines (Pazda et al., 2019). There are some groups of antibiotics which have been solely used for personal medicinal purposes in Europe, as shown in figure 1.

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4 Figure 1- Classes of antibiotics used in Europe specific to human medicine *The antibiotic group Carbapenems is part of the β-lactams class. Gathered from (Lettieri et al., 2018).

Antibiotics used for medical purposes have been categorised as either broad-spectrum or narrow spectrum. Broad-spectrum antibiotics have an extensive range of use and are used to treat various types of bacterial infections. Narrow- spectrum antibiotics targets specific bacterial infections. The consumption of broad-spectrum antibiotics has historically been higher than narrow-spectrum (Alexander, Bollmann, Seitz, & Schwartz, 2015; Jiang et al., 2014). As a result the antibiotic resistance associated with broad-spectrum antibiotics detected in conventional wastewater treatment plants have been higher than narrow-spectrum antibiotics (Hiller, Hübner, Fajnorova, Schwartz, & Drewes, 2019).

The detection of antibiotic resistance of new antibiotic have generally been within the same decade the drug was released (Lood et al., 2017). Indiscriminate antibiotic use in human treatment has led to the increasing problem of resistant pathogenic bacteria and multidrug resistance (MDR) in hospital settings (Loomba, Taneja, & Mishra, 2010; Pruden, 2014). To combat life-threatening infections caused by MDR, the world health organisation (WHO) has

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5 declared a list of last resort and emergency antibiotics. Research indicates the moderation by national-level legislation of disposal, clinical and agricultural use of antibiotics effective in reducing the global problem concerning antibiotic resistance (Pazda et al., 2019; Pruden et al., 2013). However, from the year 2000 to 2010 researchers observed an alarming increase in human consumption of las-resort antibiotics, namely carbapenems (45 %) and polymyxins (13

%) (Van Boeckel et al., 2014).

2.2 Antibiotics mode of action

The modes of action of antibiotics vary in their target areas of the bacterial cell as depicted in figure 2. The five major target areas and the classes of antibiotics which have been found to have an impact on these targets are listed in table 1. The means of action of antibiotics have shown be a hindrance of bacterial growth or cause cell lysis or death of the cell (Wennstrom Bennett, 2015, p. 1). Depending on the mentioned fate of the cells targeted, antibiotics have been classified respectively as, bacteriostatic, or bactericidal. These targets differ from the eukaryotic cell, which have made the drugs non-efficient on inherent human cells and relatively non-toxic to humans (Wright, 2010).

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6 Table 2-1 Antibiotics and their target area. Adapted from (Chander et al., 2007; Hoerr et al., 2016; Lettieri et al., 2018)

Class Example Targets

β-lactams, Glycopeptides, Fosfomycin, Bacitracin, Cycloserine, Isoniazid

Daptomycin Bacterial cell wall

Tetracyclines, Aminoglycosides Macrolides, Chloramphenicol, Clindamycin, Lincomycin

Protein synthesis (30S) Protein synthesis (50S)

Fluoroquinolones, Rifampin, Metronidazole

Ansamycins, Mupirocin, Puromycin

DNA synthesis RNA synthesis

Sulphonamides, Trimethoprim (trimethoprim- sulfamethoxazole)

Folic acid metabolism

Lipopeptides, Polymyxins Bacterial cell

membrane

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2.2.1 Inhibition of cell wall synthesis

A crucial group of antimicrobial agents have been categorised as cell wall inhibitors. Their selectivity have been based on the lack of cell walls in human and animal cells (Wright, 2010).

The primary function of the bacterial cell wall has been to provide rigidity and retain intracellular osmotic pressure to prevent cell lysis. These features are supplied by the peptidoglycan backbone (Sarkar, Yarlagadda, Ghosh, & Haldar, 2017). The standard mode of action of cell wall inhibitors is to inhibit the formation of the peptidoglycan (Ren et al., 2015, p. 20).

Some examples of antibiotics which target cell wall synthesis are β- lactams, Vancomycin, and Isoniazid. β- lactams (carpenepems, cephalosporins, monobactams and penicillins) targets the active site of the enzyme penicillin-binding protein responsible for several enzymatic reactions essential for the permeability of the peptidoglycan (Ren et al., 2015, p. 20). Isoniazid targets the cell-wall of mycobacteria by preventing the formation of mycolic acid (Anderson, Groundwater, Todd, & Worsley, 2012a, p. 327).

2.2.2 Nucleic acid synthesis inhibition

Deoxyribonucleic acid (DNA) functions as a template for genetic information. Ribonucleic acid (RNA) convert this genetic information into proteins. Nucleic acid synthesis inhibitors inhibit enzymes necessary for DNA synthesis in prokaryotes (Ren et al., 2015, p. 27). An example of RNA synthesis inhibitors mode of action is the inhibition of RNA polymerase and subsequently, the inhibition of transcription, which is the known mechanism of the antibiotic rifamycin (Anderson et al., 2012b, p. 69).

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2.2.3 Protein synthesis inhibition

The protein synthesis inhibitors target the 30S and 50S ribosomal subunits of the bacterial cell, taking advantage of the distinction between the eukaryotic 80S ribosome and the bacterial ribosomes. These agents disrupt the translation of the ribosomal protein sequence (Ren et al., 2015, p. 25). Antibiotics which target the large 50S ribosomal subunit includes macrolides clarithromycin, erythromycin and azithromycin and aminoglycosides including gentamicin and tobramycin. Tetracyclines and aminoglycosides target the 30S ribosomal subunit by binding to the sites in which tRNA would attach, inhibiting mRNA translation and preventing bacterial growth (Anderson et al., 2012c, p. 149).

2.2.4 Folic acid synthesis inhibition

Folate or Folic acid is an essential coenzyme necessary to produce nucleic acids in cells.

Production of folate occurs naturally in bacteria, contrary to mammals which need the vitamin as a part of the diet. The specificity of folic acid synthesis inhibitors like sulphonamides and trimethoprim to bacterial cells have been to target the reactions of folate synthesis (Ren et al., 2015, p. 29). Sulphonamides have shown to inhibit dihydrofolate synthesis by inhibition of the enzyme dihydropteroate synthetase, while trimethoprim stops the reaction by inhibiting the enzyme dihydrofolate reductase (Sköld, 2011).

2.2.5 Cell membrane inhibition

The last mode of action is the cell membrane inhibition, the mechanism of Lipopeptides such as daptomycin and Polymyxins such as colistin. These agents act non-selectively on both the outer and inner cell membrane, causing the loss of function of the membrane (Ren et al., 2015, p. 21). These actions include the disrupting the shape of the membrane, altering energy pumps binding onto sections of the membrane.

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9 Figure 2 - Antibiotic targets and bacterial mechanisms of resistance. Gathered from (Wright, 2010).

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2.3 Antibiotic resistance in bacteria

The ability of the bacterial cell to grow when subjected to an antibiotic is regarded as antibiotic resistance (Luby, Ibekwe, Zilles, & Pruden, 2016). Evidence of the increasing presence of antibiotic resistance in pathogenic bacteria has been attributed to extensive anthropogenic use and release of antibiotics into the biosphere over the last century (Pruden, 2014). Long term exposure to antibiotics exerts selective pressure on bacteria leading to the more rapid evolution of resistant mechanisms (Wright, 2010). Bacteria possess the ability to develop resistant mechanisms towards antibiotics, expressed by their ARGs (Lin et al., 2015). The authors further showed that resistance could occur naturally (intrinsic) or because of spontaneous mutations (de novo) in bacteria. The vertical transfer of genetic material from parent to offspring is performed by transmission of the information located on the bacterial chromosome (Pazda et al., 2019). Lastly, antibiotic resistance has shown to be transferred laterally by horizontal gene transfer (HGT) in several ways, as shown in figure 3 (Blakely, 2015). ARG transfer has shown to not necessarily be restricted to specific species or genera because of HGT by MGEs, and these transfer methods have been considered the most important modes of ARG spreading between bacteria (Muniesa, Colomer-Lluch, & Jofre, 2013).

Figure 3 - Mechanisms of vertical and lateral gene transfer in bacteria. Gathered from (Blakely, 2015).

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2.3.1 Horizontal gene transfer

HGT occurs via transformation, conjugation or transduction (Aminov, 2011). The mechanisms mentioned include, respectively, uptake of exogenous genes from the environment by the bacteria, transfer of genes between bacterial cells by direct contact and the transfer of genes through the medium of bacteriophages or other MGEs (Pazda et al., 2019). MGEs have shown to mediate the transfer of the bacterial genetic material within the cell or between distinct cells.

Plasmids, transposons and integrons are MGEs of importance for HGT (Rizzo et al., 2013).

Plasmids are small, most often circular, DNA containing segments which have been found to be present in all bacterial cells (Snyder & Champness, 2007b, p. 197). Plasmids have shown to exist separately from the bacterial chromosome and self-replicate independently during cell division (Frost, Leplae, Summers, & Toussaint, 2005). Plasmids have not shown to contain genes that encode life-essential mechanism for the bacteria. However, many carry genes coding resistance to heavy metals and antibiotics (Bennett, 2009). These plasmids have been named resistance plasmids. Resistance plasmids can be self-transmissible or mobilisable, in which they respectively carry and transfer genes or carry resistant genes and is transferrable by the self- transmissible plasmids (Snyder & Champness, 2007d, p. 425). Transposons move DNA sequences within the genome, although some transposons have been known to be conjugative and thus able to transfer resistant genes to other bacterial cells (Snyder & Champness, 2007d, p. 424). Mobile integrons have shown to put together open reading frames and carry out site- specific recombination of exogenous DNA material in gene cassettes (Mazel, 2006). The intrgrons have been shown to have the ability to integrate into plasmids and transposons (Rizzo et al., 2013). Integrons have thus shown to be of importance for antibiotic resistance dissemination (Xue et al., 2019).

HGT by conjugation is the mechanism most abundantly described in the literature (Lood et al., 2017). During conjugation, DNA transfer occur from a donor cell by a conjugative MGE (e.g.

plasmid, transposon) by direct contact between bacterial cells through a pilus or adhesin on the bacterial surface (Rogers & J. Kadner, 2019). Both plasmid DNA and chromosomal DNA can be transferred by plasmids during this process (Snyder & Champness, 2007a, p. 263). Plasmids, which inherently cannot exist in the same bacterial cell if they have similar replication mechanisms, have been categorised as incompatible (Frost, Leplae, Summers, & Toussaint, 2005).

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12 Moreover, conjugation has shown to occur by transposons which, for example, have been discussed to be a possible explanation of the wide-spread occurrence of the tetracycline resistance encoding gene tetM in the pathogen Enterococcus faecalis (Snyder & Champness, 2007a, p. 271). Conjugation has previously been considered the most critical mechanism for the spread of ARGs. However, recently the mechanisms of transformation and transduction have gotten more attention in studies concerning the environmental reservoir of ARGs (resistome) (von Wintersdorff et al., 2016).

Transformation is the uptake of free extracellular naked DNA fragments by bacteria which are competent to bind these fragments to their genome or incorporate as a plasmid (Rogers & J.

Kadner, 2019). Competence has shown to be induced by chemical or heat treatment. However, natural competence (the ability a bacteria has to receive or donate DNA to other cells by transformation) does not occur in all bacteria (Snyder & Champness, 2007c, p. 278). WWTPs provides a vast reservoir of naked DNA fragments, and thus provide a suitable environment for transformation especially since the presence of antibiotics have been seen to stimulate the transformation of ARGs (von Wintersdorff et al., 2016). However, the DNA degrading enzyme DNAse exist in wastewater and have shown to limit the transformation potential (Bitton, 2011, p. 16).

Bacteriophages (viruses using bacteria as hosts) have shown to facilitate the transfer of small DNA fragments between different bacteria by uptake of DNA into the phage, followed by injection of donor cell genetic material into a new host (Bitton, 2011, p. 17). Transduction by bacteriophages has for a long time been regarded as an insignificant mode of gene transfer, due to its limitations of inefficiency and rare cases if interspecies transfer (Rogers & J. Kadner, 2019). Although, recent environmental studies using metagenomic and qPCR methods have increased the interest in this method as influential on the resistome in compartments such as wastewater and sludge (von Wintersdorff et al., 2016). Bacteriophage transduction importance in WWTPs was pointed out by Lood et al., (2017). The authors expressed that viruses have shown higher survivability during wastewater treatment processes than bacteria. The high density of viruses and the chemical stressors in the wastewater have shown to induce the transduction rates of bacteriophages (Aminov, 2011).

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2.3.2 Mechanisms of resistance

There have been detected many different mechanisms for antibiotic resistance expressed in bacterial cells (Byarugaba, 2010). Multiple mechanisms to prevent the impact of an antibiotic have been found to occur in single bacterial cells (Lin et al., 2015). These mechanisms include reduction of the drugs ability to penetrate through the membrane of the cell wall, modification of binding target of the antibiotic (modification), the production of antibiotic degrading enzymes (degradation) and lastly repulsion of the antibiotics through pumps (efflux) (Lin et al., 2015).

The strategy of modifying cell wall permeability has mostly been linked to gram-negative bacteria (Munita & Arias, 2016). The process involves modification of the porin channel properties, to avoid passage of the antimicrobial drugs into the bacterial cells through the cell wall, a resistance trait common for the antibiotics β-lactams and fluoroquinolones (Pagès, James, & Winterhalter, 2008). Production of antibiotic degrading enzymes have shown to enable bacteria to degrade components of the antimicrobial agents, thus inactivating the bactericidal or bacteriostatic properties of the antibiotic (Lettieri et al., 2018). This mechanism has been observed in resistance to Tetracyclines, coded by the gene tet X (Aminov et al., 2001).

Modification of binding target to avoid the effects of antimicrobial drugs in the bacterial cells include changes in ribosomal proteins, 16s rRNA, DNA gyrase subunits, penicillin-binding proteins and cell wall precursor components (Lettieri et al., 2018). Macrolide resistant bacteria create methylase to methylate the antibiotic target site 23srRNA (a part of 50S) (Leclercq, 2002), which is coded by erm genes (Pazda et al., 2019). Bacteria which portray β- lactam resistance use mainly the β- lactamase enzymes to destruct the antibiotic compound by cleaving the β-lactam ring responsible for the antibacterial actions of these compounds (Lin et al., 2015).

These degradation enzymes are encoded by amp, bla and oxa genes (Pazda et al., 2019).

Extruding antimicrobial agents through the means of efflux pumps is a method that has been used by bacterial cells to remove antimicrobial drugs once it has entered the cell walls using specific or nonspecific membrane proteins. 23 tet genes encoding Tetracycline resistance by efflux pumps have been detected (Munita & Arias, 2016). Macrolide efflux pumps of the ATP- binding cassette type is encoded by mrs(A) gene (Leclercq, 2002). Some bacteria have shown to operate multidrug efflux pumps, making them able to extrude several types of antibiotics and other pharmaceuticals (Nikaido, 2009).

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2.4 Wastewater treatment

Wastewater treatment has been commonly defined as mechanical, chemical or biological applications that are used to make sure the effluent released to the receiving surface waters is safe and free of contaminants (Park, 2007). The treatment of wastewater is most often divided into the categories of preliminary, primary, secondary and tertiary treatment (Tchobanoglous et al., 2003, p. 12). Preliminary treatment involves the removal of objects of significant size, including sticks and grit that inflicts the operational equipment of the WWTPs. The further step of primary treatment is the physical removal of suspended solids, in addition to fat or grease removal from the surface. Secondary treatment involves chemical and biological removal of suspended solids, organic and inorganic matter through conventional processes like activated sludge (AS), trickling filters and oxidation ponds. Secondary treatment can also involve the use of aerobic, anoxic, or anaerobic digestion by re-circulation and occasionally disinfection (Pei et al., 2019). Additional processes for reducing organics, metals, nitrogen, phosphorous, recalcitrant chemicals and pathogens like additional disinfection, coagulation, flocculation and filtration are categorised as tertiary treatment processes (Gerba & Pepper, 2015).

The presence of emerging pollutants in WWTPs has become more apparent. The previous goal to avoid surface water eutrophication and release of pathogens have been proposed to be extended to include environmental persistent pharmaceutical pollutants (EPPP) chemicals, such as antibiotics (Henze, van Loosdrecht, Ekama, & Brdjanovic, 2008). Antibiotics and ARG have been considered emerging contaminants (Al-Riyami et al., 2018). The WWTPs purpose as a barrier of pollutants to the receiving environment have been under recent evaluation due to the amounts of antibiotics and ARGs in effluents of WWTPs and the lack of regulation concerning these compounds (Manaia et al., 2018).

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2.4.1 Antibiotics in WWTPs

The occurrence of antibiotics in influent water in wastewater treatment plants (WWTPs) have shown to originate from several sources including medical waste from hospitals and pharmaceutical industries, municipal sewers and agricultural runoff (Burch et al., 2019). Runoff from agriculture consists of non-therapeutic antimicrobial agents used for growth purposes in the animal husbandry. Hospital effluent and municipal sewage contain large amounts of antimicrobial waste containing parent and partly metabolised medicinal antimicrobial agents which have been secreted through the faecal route of humans or animals (Huang et al., 2013).

Resistance detected in clinical setting have shown to originate from human consumption but also environmental compartments because of antibiotic release from WWTPs, medical waste and manure from agriculture as shown in figure 4 (Lood et al., 2017; Qiao, Ying, Singer, &

Zhu, 2018).

Figure 4 – Route of antibiotics from human and animal consumption through WWTPs to the environment and the resulting resistance problem in clinical settings. Adapted from (Qiao et al., 2018)

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16 Antibiotic load in wastewater influent have shown to vary by region. In a study that summarised the values of antibiotics in WWTPs in China reported that the most abundant antibiotics detected in WWTPs were sulphonamides, tetracyclines and fluoroquinolones (Qiao et al., 2018). The authors further expressed the high variations in inlet concentrations, from a few ng/L up to tenfold µg/L. Moreover, the values detected in influent and effluent samples did not always correlate well with values from other countries. Most wastewater treatment plants have not been designed to remove micropollutants, such as antibiotics (Kurt et al., 2017). Antibiotics have been detected in effluents from WWTPs worldwide with mean concentration values ranging from below 0.5 µg/L to over 5 µg/L, depending on the type of antibiotic and treatment facility (Lettieri et al., 2018).

Antibiotics tend to bioaccumulate (Arslan-Alaton & Dogruel, 2004). The compounds have been known to persist in aquatic environments and interfere in wastewater treatment plants by selective inhibition of active microorganisms (Abbassi, Abusaleem, Zytner, Gharabaghi, &

Rudra, 2016). Antibiotics have been categorised as organic micropollutants (OMPs), and increasing interest in research of removal of these pollutants has been seen in recent years (Harb

& Hong, 2017a).

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2.4.2 Antibiotic resistance in Wastewater treatment plants (WWTPs)

Indications of resistance to antibiotics, namely ARBs and ARGs, has frequently been detected in all stages of various wastewater treatment facilities (Pang, Huang, Xi, Hu, & Zhu, 2016). 3, 15, 18, 38 and 86 different types of genes coding for resistance to sulphonamides, quinolones, macrolides, tetracyclines and β- lactams have respectively been detected in conventional treatment plants, in addition to 26 different genes coding for multidrug efflux pump resistance (Pazda et al., 2019). Genes coding for resistance to tetracyclines have been detected most frequently and abundantly. Genes coding for resistance to sulphonamides has also been highly detected in WWTPs (Xue et al., 2019). The sul 1 gene have been most abundant in various WWTPs (Du et al., 2015).

As described by Manaia et al. (2018), WWTPs have become a threat to human and animal health because of the favourable conditions for horizontal gene transfer and the proliferation of ARGs. WWTPs have been regarded as hotspots for the spread of ARB and ARGs (Rizzo et al., 2013). The density of microorganisms, the proximity of bacterial cells and the nutrient-rich environment in the WWTPs have shown to promote growth and cell division in addition to lateral (horizontal) gene transfer (Pazda et al., 2019).

Another contributing factor has been the amount of partly metabolised or mother substance of antibiotics that is transferred through the inlets of treatment plants. Antibiotic residues at subinhibitory concentrations have been reported to cause selective pressure, resulting in antibiotic- resistant bacteria (ARB) increase in relative abundance to non-resistant bacteria which is sensitive to antibiotics (Birošová et al., 2014). Other pollutants, such as heavy metals, have been shown to enhance the propagation of efflux ARGs in wastewater (Mao et al., 2015). Co- selection of resistant efflux mechanisms displayed by bacteria to both antibiotics and heavy metals have been shown to occur in WWTPs (Lettieri et al., 2018). A metagenomic study by Li, Li, & Zhang (2015) on plasmids showed similar patterns of ARGs and metal resistant genes in WWTP influent.

Mobile genetic elements have become targets of recent research regarding antibiotic resistance (Lood et al., 2017). Their role in HGT as recipients, donors, and carriers of ARGs has shown to affect the fate of ARGs in WWTPs (Tong et al., 2019). For example, class 1 integrons used as targets for studying antibiotic resistance proliferation in wastewater have been shown to indicate the potential intercellular transfer of ARGs (Jilu Wang, Mao, Mu, & Luo, 2015).

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2.5 Tools for assessing antibiotic resistance in WWTPs

The methods used for the detection and surveillance of ARGs and ARBs in WWTPs have mainly been divided into molecular-based (culture-independent) approaches or culture-based approaches. Recent research has also included metagenomic methods (Rizzo et al., 2013). The culture-based methods focus on the targeted isolation of clinical pathogens relevant to human health (Do, Murphy, & Walsh, 2017). Culture-independent methods focus on targeted direct analysis of nucleic acids and can give indications of antibiotic-resistant potential in the microbial community based on singular detection or quantification of ARGs in the wastewater matrix by PCR methods (Manaia et al., 2018; Rocha et al., 2019). Metagenomic high throughput sequencing methods offer a non-targeted broader overview of the gene pool present in wastewater samples (Manaia et al., 2018).

2.5.1 Culture-based methods

The most convenient and accessible methods for determining antibiotic susceptibility have been culture-based approaches (Rizzo et al., 2013). The basis of these methods has relied on the evaluation of microbial growth when exposed to antibiotics. These methods have been performed on isolated bacterial strains of clinical concern (e.g. Enterococcus, Salmonella and Staphylococcus) on growth media with varying degree of selectivity (McLain, Cytryn, Durso,

& Young, 2016). The culture-based approaches have shown to have valuable advantages in assessing antibiotic resistance in wastewater samples, such as describing the phenotype (bacterial traits) of bacterial isolates (Manaia et al., 2018). Minimum inhibitory concentrations (MIC) of antibiotics have been determined by culture-based approaches (Selvaraj et al., 2018), which can be valuable in making regulations for antibiotics threshold values in WWTPs based on the predicted promotion effect on resistance in the ecosystem (Bengtsson-Palme & Larsson, 2016).

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2.5.1.1 Agar dilution

Antibiotic susceptibility testing by agar dilution has been utilised to determine MIC in bacterial isolates (Do et al., 2017). Bacteria in standardised suspension have been transferred onto agar plates with different antibiotic concentrations to determine the threshold level at which the bacteria do not grow on the medium.

2.5.1.2 Broth dilution method

MIC of antibiotics have been determined by broth dilution methods (Martineau et al., 2000).

The method involves either microdilution or microdilution tests. Macrodilution test has determined the MIC of clinical pathogens by incubating them in tubes with growth medium having an increasing 1:2 gradient concentration of antibiotics at 37°C. After incubation, MIC has been determined based on turbidity (indicating bacterial growth) in the tubes by the use of a spectrophotometer (Do et a., 2017). A more economical and time-saving version of this method has been developed, namely the microdilution method. Well-plates with 96 or 384 wells containing 1:2 dilutions in the same sense as the macrodilution method has provides an opportunity to test more antibiotics at once (Do et al., 2017). 290 isolates from the influent and effluent of a wastewater treatment plant in Saudi Arabia was tested by microdilution method do determine antibiotic resistance in bacterial isolates (Al-Jassim, Ansari, Harb, & Hong, 2015).

2.5.1.3 Disc diffusion method

The disc diffusion method often referred to as Kirby Bauer, determines the specific isolate susceptibility to the tested antibiotic by using a spread plate of a bacterial isolate on specific growth medium. An antibiotic-soaked disc is placed on growth medium (agar plate), and the susceptibility is estimated by the diameter of the ring formed around it after incubation (Do et al., 2017). The ring indicates a lack of growth outside the antibiotic disc, called the zone of inhibition. Based on a standard interpretation charts, the antibiotic in question has been qualitatively categorised as resistant, intermediately susceptible or susceptible to the type of antibiotic in question. Disc diffusion is the susceptibility test which has required least equipment cost and operational skills (Do et al., 2017).

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2.5.1.4 Etest

A simplified test to detect the minimal MIC have been shown to be the Etest which was introduced in 1988 by AB Biodisk in Sweden (Picard, 1990). The principle of this method is a strip containing a gradient of antibiotic concentration. The strip is placed on bacterial inoculated agar plates. After incubation, the MIC has been determined based on the intersection of the bacterial growth with the strip, depicted in an elliptical shape, as shown in figure 4 (Jorgensen

& Ferraro, 2009). The method has been used in wastewater treatment, but due to expensive test strips, multiple antibiotics testing by this method have not been preferred (Do et al., 2017).

Figure 5 – Etest antibiotic susceptibility test. Gathered from (Jorgensen & Ferraro, 2009).

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2.5.2 Culture-independent methods

Several culture-independent or molecular methods for detection and quantification of ARGs in wastewater has been developed and optimised during recent years. The methods target nucleic acids or proteins of the bacterial cell (Luby et al., 2016). Molecular tools which have been developed for ARG assessment in environmental samples are polymerase chain reaction (PCR), quantitative PCR (qPCR), DNA hybridisation and DNA microarray. The most commonly used and proven methods for wastewater analysis are PCR and quantitative qPCR (Zhang et al., 2009).

2.5.2.1 Polymerase Chain Reaction (PCR)

Antibiotic resistance is genetically encoded (Pazda et al., 2019). The presence of specific genes coding for antibiotic resistance have been qualitatively analysed by polymerase chain reaction (PCR). After the discovery of the method in 1983, a paradigm shift has occurred in modern life sciences (Pillai & McKelvey, 2016, p. 65). The method has allowed amplification of a specific section of extracted DNA (target sequence) to a size which can be visualised by gel electrophoresis (Luby et al., 2016). The amplification (copying) of genes have been enabled by the use of a mix of chemical reagents and a DNA template (extracted from the sample) to mimic the natural DNA duplication process (Bassin, Dezotti, & Rosado, 2018). The reaction has been performed by repeatedly heating and cooling in an automated thermal cycler to denature DNA, followed by annealing using forward and reverse primers and a thermostable DNA polymerase.

Furthermore, DNA elongation occur at final stage, creating a new DNA strand of the target region (Pillai & McKelvey, 2016, p. 66). Polymerase chain reaction (PCR) has been valued as a reliable, effective and sensitive technique for amplification and detection of specific segments of DNA, such as single ARG analysis or multiple gene analysis in the form of multiplex PCR and high throughput PCR array (Ng et al., 2001; Zhu et al., 2013). Traditional PCR is a qualitative method. For ARG detection in wastewater samples, PCR has respectively been shown to yield highly consistent results with high sensitivity (Rocha et al., 2019).

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2.5.2.2 quantitative PCR (qPCR)

Quantification of ARGs present in wastewater have been performed by quantitative PCR (qPCR). qPCR follow the same preliminary principles as PCR followed by an additional DNA probe method or dye-based method used to detect fluorescence in real time during the thermal cycling (Volkmann, Schwartz, Kirchen, Stofer, & Obst, 2007). Signals have been plotted against standard curves made prior and extrapolation of signals from unknown samples have resulted in quantification of target genes. The method have not detected viability or functional abilities of host enharbouring the selected ARGs, it has only indicated presence and quantity of the targeted genes in the sample matrix or an isolated organism (Diehl & LaPara, 2010).

Moreover, qPCR have been used to evaluate conjugation and transduction potential in samples by studying marker genes of incompatibility groups in plasmids and the genes recovered from isolation and nucleic extraction of bacteriophages (Luby et al., 2016). qPCR has been used in numerous studies of wastewater samples, contributing to the large amount of data currently gathered on resistant genes in WWTPs (Du et al., 2015; Mao et al., 2015; Pärnänen et al., 2019;

Jilu Wang et al., 2015).

2.5.2.3 Viability methods

PMA- qPCR: qPCR combined with propidium monoazide (PMA) treatment have shown to successfully quantify viable cells in wastewater samples (Li et al., 2014). The method have been based on exclusion of cells which do not contain intact membranes by the use of intercalating dyes in PCR (Nocker, Sossa-Fernandez, Burr, & Camper, 2007). PMA inhibits the amplification of DNA from dead cells which disables the amplification product of DNA in non- viable cells (Fittipaldi, Codony, Adrados, Camper, & Morató, 2011).

Flow cytometry: Similarly, to the PMA- method flow cytometry has detected live or dead cells based on membrane integrity. This method has shown to detect viability by a monochromatic light source on bacterial cells which scatters light and fluoresce due to impermeant cell staining (Safford & Bischel, 2019). Moreover, flow cytometry has been used to monitor HGT events in activated sludge (Pei & Gunsch, 2009). However, this method has shown to portray some underestimated results due to cell clumping in the cytometer (Nocker et al., 2007).

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2.5.3 Metagenomics

Metagenomic analysis of antibiotic resistance in the form of shotgun sequencing and high throughput sequencing has been successful in making overview in the abundance of ARG in WWTP matrices and other environmental samples (von Wintersdorff et al., 2016). Descriptive metagenomics have relied on direct extraction of DNA from all microorganisms in the sample environment, followed by sequencing and comparison to databases containing known ARG or protein sequences (Yang & Zhang, 2017). These methods have been beneficial due to the exclusion of the biased results from targeting isolated microorganisms in culture-based antibiotic resistance studies (von Wintersdorff et al., 2016). Additionally, the biased results from manual errors, primer selection or amplification in PCR and qPCR have been avoided (Yang, Li, Ju, & Zhang, 2013). The restriction of the metagenomic methods for detection of ARG in wastewater systems has been the inability to detect low abundance genes (Pärnänen et al., 2019). The reliability and accurateness of the databases aligning the sequencing data have been determined as a crucial component the use of this method for antibiotic resistance research.

(Yang et al., 2013). Metagenomics studies have been performed in recent studies to analyse ARGs and MGEs in various WWTPs (Li et al., 2015; Yang et al., 2013).

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2.6 Removal of antibiotic resistance determinants in WWTPs

Due to large deposits of antimicrobials and ARGs in wastewater, actively targeting antibiotic resistance determinants (ARG, ARB) in current wastewater treatments plants and reducing the ARG potential released into the environment by optimising current treatment methods have been considered as an opportunity to combat resistance issues (Diehl & LaPara, 2010). The antibiotic substances or determinants of resistance are currently unregulated in wastewater effluents (Pazda et al., 2019). However, the spread of contaminants like ARBs and ARGs in the environment from WWTPs have shown to be the primary source of impact on environmental compartments by the spread of resistance in the microbiome (Karkman et al., 2016). The impact of antibiotics WWTP effluent release in receiving waters as a cause of selective pressure are also important to consider. Studies comparing rivers upstream and downstream of WWTPs have shown significantly higher concentrations of antibiotics downstream of treatment plants (Rodriguez-Mozaz et al., 2015; J. Xu et al., 2015).

Current wastewater treatment processes implemented have shown to successfully reduce large amounts of microorganisms (bacteria, viruses, helminths), including antibiotic resistant bacteria (ARB), but have not shown the same effectiveness at removing ARGs (Pazda et al., 2019).

Because of the lateral gene transfer potential of bacteria, the inactivation and removal of ARBs and ARGs in wastewater treatment facilities have respectively been considered essential to stop the spread of antibiotic resistance in the WWTPs processes (Pang et al., 2016). Additionally, the removal have shown to be vital to reduce their further spread into the receiving environmental microbiome (Berendonk et al., 2015; Pruden, 2014). The removal efficiencies have been reported to vary significantly by plant design and operation (Barancheshme & Munir, 2019; Munir, Wong, & Xagoraraki, 2011; Pazda et al., 2019). ARG abundance and variations of specific genes present in the wastewater matrices have also been discussed to impact the removal potential of the individual treatment plants (Xue et al., 2019). In recent years there has been an increase in research in the applications of conventional and advanced WWTPs and how the different processes affect the removal of ARGs (Ghernaout, 2020).

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2.6.1 Conventional treatment

Most of the research on antibiotic resistance in wastewater has been on studies in conventional treatment processes (Pazda et al., 2019). Conventional treatment processes include physical, chemical, and biological treatment processes, albeit biological treatment processes have been the most implemented process in WWTPs worldwide (Xue et al., 2019).

2.6.1.1 Activated sludge

Conventional activated sludge (CAS) treatment is regarded as the most common suspended growth treatment process (Pazda et al., 2019). This biological treatment process involves the activation of microorganisms able to remove carbonaceous biological oxygen demand and nutrients by aeration, the formation of settleable flocks and re-circulation of active biomass (Tchobanoglous et al., 2003, p. 557). The microbial population of CAS consist of mostly bacteria (95 %) (Jenkins, Richard, Daigger, & Jenkins, 2004). The operational temperatures are commonly between 10- 25 °C range (Miller, Novak, Knocke, & Pruden, 2016).

The conventional biological treatment methods have shown different removal rates of pharmaceuticals, such as antibiotics. Some have shown less than 20 % removal while others have shown more than 90 % removal of antibiotics (Kurt et al., 2017). Antibiotics have not shown to degrade efficiently in the environment of the CAS due to short solid retention times (Michael et al., 2013). During activated sludge processes antibiotics and other organic micropollutants have been shown to accumulate in the sludge due to sorption onto solids in the reactors. especially hydrophobic antibiotics (Hollender et al., 2009).

The enrichment of ARGs in sludge have been shown to be favourable in conventional biological treatment plants like the CAS process (Lupo, Coyne, & Berendonk, 2012; Michael et al., 2013).

The combination of high bacterial density, the proximity of cells and antibiotics (even at subinhibitory levels) favours the transfer of ARGs by HTG (Wang et al., 2015). The re- circulation of sludge into the reactors provide a better environment for HGT. HGT have shown to be further induced by aeration (Tong et al., 2019).

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26 Two main products result from CAS wastewater treatment, treated effluent water and biosolids.

The fate of ARGs removed during the CAS process have implications for the reuse of these WWTP products, illustrated in figure 4 (Harb & Hong, 2017a). Biosolids have been described by Lu et al., (2012) as the treated product of sludge, rich in organics and nutrients. Bacteria settling in primary or secondary clarifiers is the primary removal mechanism of ARGs in CAS (Chen & Zhang, 2013). The utmost (>99%) of ARB harbouring ARGs removed in activated sludge processes have shown to end up in the sludge (Burch, Sadowsky, & LaPara, 2013a;

Calero-Cáceres et al., 2014).

Figure 6 - Fate of antibiotic resistance in conventional activated sludge treatment processes.

Gathered from (Harb & Hong, 2017a).

Reports of ARG removal in CAS have been conducted. Removal efficiencies of ARG in studies of CAS effluent and raw influent water in China and Michigan was 89-99.8 % and 2.40-4.60 log removal respectively (Mao et al., 2015; Munir et al., 2011). Others have reported a 1-3 order of magnitude removal (Chen & Zhang, 2013). Sludge analysis of CAS plants in China showed ARG dewatered sludge discharge of 6.1 ± 0.5 x ¹⁹ copies/day. Also, 10 of the 23 tested ARGs showed enriched values in the effluent water and sludge compared to the influent. Authors have raised question about the hazardous implication of using biosolids from CAS plants for land application purposes due to the probability of ARGs transferring into the soil microbiome (Chen

& Zhang, 2013; Munir et al., 2011). The release of ARGs through biosolids have shown to be 1000 times higher than effluent water (Munir et al., 2011).

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2.6.1.2 Sludge treatment

Sludge treatment methods have, in recent years, been considered as a critical part of WWTP in ARG removal strategies to minimise environmental impact (Burch et al., 2013b; Xue et al., 2019). Sludge treatment methods usually include thickening, digestion and dewatering processes (Tchobanoglous et al., 2003, p. 1453). The original purpose of sludge treatment have been to stabilise the sludge to prevent odour and reduce its volume to decrease the cost of storage and further handling (Ambulkar & Nathanson, 2019). Substantial amounts of ARGs have been detected in untreated sludge from biological WWTPs (Diehl & LaPara, 2010).

Standard sludge treatment includes digestion (aerobic or anaerobic) or composting. Limited studies performed on aerobic digestion have shown some degree of removal of ARGs from wastewater biosolids, although the method has shown less effective than composting and anaerobic digestion (AD) (Burch et al., 2013b; Diehl & LaPara, 2010).

2.6.1.2.1 Anaerobic digestion

Most current studies on ARG removal in sludge treatment processes have been conducted using the anaerobic digestion (AD) (Xue et al., 2019). The removal mechanisms of ARGs in AD have been attributed to the destruction of extracellular DNA by hydrolysis and biodegradation (Ma et al., 2011). Several key operating parameters have been shown to affect the performance of anaerobic digestive ARG removal in sludge, including temperature, level of pre-treatment and types of ARGs present in the sludge (Xue et al., 2019).

ARG removal in anaerobic digestion have been affected by temperature. Studies on AD have demonstrated that mesophilic (~35°C) digestion is inferior to thermophilic digestion (>50°C) for the efficiency of ARG removal (Ghosh, Ramsden, & LaPara, 2009; Yang, Li, Zou, Fang, &

Zhang, 2014). Correlations between increased temperature and ARG removal efficiency have been shown up to 60°C, although no improvement beyond these temperatures have been seen (Burch, Sadowsky, & LaPara, 2016; Diehl & LaPara, 2010; Ma et al., 2011). Additionally, the survival of ARB in mesophilic AD has shown to be higher than thermophilic (Miller et al., 2016). A possible explanation has been given by Tian, Zhang, Yu, & Yang (2016) to be reduction in the diversity of the microbial populations due to the high temperatures.

Metagenomic study by Tian et al., (2016) on the treatment of sludge by AD during thermophilic temperatures showed higher reduction values of the overall resistome and the HTG related

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28 mobilome (integrons, plasmids and insertion sequences) compared to digestion under mesophilic temperatures. Significant reduction of MGEs was shown in thermophilic AD treatment and thus the method was deemed beneficial to decrease in the proliferation potential of ARGs in biosolid application. The relative abundance of ARGs decreased by 64.99 % in thermophilic and 46.53 % in mesophilic AD.

Improved ARG removal has been shown in AD combined with pre-treatment (Ma et al., 2011;

Pei, Yao, Wang, Ren, & Yu, 2016; Tong et al., 2016, 2017). In the study by Wang, Li, & Zhao (2019), the ultrasonic pre-treatment method was the most efficient in ARG reduction, compared to thermal hydrolysis or chemical treatment. Ultrasonic pre-treatment enhanced ARG removal from 50.77% (AD without pre-treatment) to 75.07% (Wang et al., 2019). However, ARG rebounding has been seen to occur after thermal hydrolysis pre-treatment (Ma et al., 2011;

Wang et al., 2019). In the study by Wang et al. (2019) the ultrasonic pre-treatment method was the most efficient in ARG reduction, compared to thermal hydrolysis or chemical treatment.

The ultrasonic pre-treatment enhanced ARG removal from 50.77 % (AD without pre-treatment) to 75.07 % .

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1.6.1.3 Constructed wetlands

Constructed wetlands (CW) are human-engineered semi-aquatic ecosystems made to simulate the processes occurring in natural wetlands (Barancheshme & Munir, 2018). The treatment method uses vegetation and acts as a biofilter. It is a well-documented treatment for the removal of microorganisms, nutrients (nitrogen and phosphorous) and organics thus decreasing the biological and chemical oxygen demand (Li et al., 2014;Wang et al., 2013). Phosphorus removal has however shown to be limited in saturated CW vegetation (Sharma et al., 2016).

Bacterial pathogen removal by constructed wetlands has been reviewed by the global water pathogen project to range from 1 log10 to 3 log10 reductions (University of Ouagadougou et al., 2019). Constructed wetlands have been categorised according to the level of submersion of the given vegetation (macrophyte) or according to the flow patterns (Vymazal, 2010). The three main models of flow patterns are free surface-flow constructed wetland (FSF-CW), vertical subsurface flow constructed wetland (VSSF-CWs) and the horizontal subsurface flow constructed wetland (HSSF-CW) (Huang et al., 2015).

The purpose of CW has predominantly been to remove harmful components in runoff from agriculture and animal farms. However, it has also been implemented to treat numerous types of wastewater and stormwater (Vymazal, 2010). The extensive use of antibiotics in growth promotion and their implications of these antibiotics in antibiotic resistance have increased scientific focus on constructed wetlands in agricultural areas for ARG removal (Sharma et al., 2016). As stated by Huang et al., (2015), the agricultural runoff wastewater implementations need improvement to prevent the spread of antimicrobial resistance originating from farm animals and fertilisers.

Pollutant removal in CWs depends on processes showed in figure 4, namely substrate adsorption, photolysis, volatilisation, plant uptake and biodegradation (Chen et al., 2016). ARG removal by CW has been mostly linked to soil sorption and biodegradation (Chen et al., 2015).

The types of microorganisms (mostly bacteria) responsible for the degradation have shown to be diverse (Fernandes et al., 2015). Moreover, the bacterial structural community of CW have been regarded as most important for enhanced antibiotic removal (Liu et al., 2019). The bacterial degradation have shown to account for 94% of antibiotic removal in CWs (Chen et al., 2019; Fernandes et al., 2015).

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30 Figure 7 - Removal mechanisms of pollutants in constructed wetlands (Sharma et al., 2016)

Tetracyclines are commonly found in wastewater from swine farms. Studies have been conducted to confirming the presence of several types of tetracyclines in both the influent and effluent from conventional wastewater plants or lagoons receiving swine manure, along with an increase in the relative abundance of ARG (tet genes) in wastewater lagoons from the influent manure (Barkovskii & Bridges, 2012; Chen et al., 2012; Cheng et al., 2013). A study by Huang et al., (2015) was conducted on the removal of the antibiotics tetracycline, oxytetracycline and chlortetracycline and some correlating resistant genes, tetA, int1 and tet W, O, M, X respectively in a vertical up-flow constructed wetland (VUF-CW). This study showed removal efficiencies from between 45.4 % and 99.9 % of most tet genes, although some genes (tetO, tetM, tetX) showed to have a slight increase during the process.

Hydraulic flow patterns have shown to affect the potential removal of ARGs and antibiotics (Karaolia, Michael, & Fatta-Kassinos, 2017a). The oxygenation of the vertical flow CW systems has shown to induce nitrification, which in turn have provided better conditions for antibiotic degradation (Zhi, Yuan, Ji, & He, 2015). Oxygen available around the plant roots have been shown to enhance the microbial structure and thus create a better environment for microbial degradation of pollutants (Sharma et al., 2016).

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31 Subsurface flow have shown to have better ARG removal because of the filtration ability of have shown to be higher (Huang et al., 2017). Antibiotic removal was shown by Berglund et al., (2014) in a full-scale surface flow CW in Sweden to be effective. However, no significant change in ARG was seen. A mesocosm study by Chen et al., (2016) studying 6 different CW configurations showed highest removal rates using VSSF-CW all with 80-99 % removal for tested ARG (tetO, tetM, tetX, tetG, sul(1-3), erm(B, C), cmlA). In other studies conducted using VSSF-CW and vertical upflow CW (VUF-CW), ARGs were not successfully removed (Chen et al., 2015; Liu et al., 2014).

Combined and integrated systems have shown to be of potential for ARG reduction. An integrated CW (ICW) consisting of a series of all the flow regimes showed removal of 1-3 orders of magnitude of all ARGs tested in this study (Chen et al., 2015). The exception was ermC with a removal of 43 %, the rest of the genes (sul1, sul2, tetM, tetO) tested had an 83 - 100 % removal efficiency during the treatment. This combined configuration was notably also efficient in antibiotic removal. Hybrid CW systems studied by Chen et al., (2019) showed good removal of organics and nutrients and ARGs, with a 87.8 - 99.1 % removal of all target ARGs.

The authors concluded VSSF with artificial aeration combined with HSSF without aeration proved to be the most efficient for the removal of ARGs and other emerging organic pollutants.

The upsides of using constructed wetlands as the only wastewater treatment implementation compared to other conventional treatment methods are the operational and energy maintenance costs, which are 2-10 times lower compared to other conventional wastewater treatment processes (Vymazal, 2010). CWs have shown to be more efficient in ARG removal than other conventional WWTPs (García et al., 2020), although complete removal has yet to be observed (Karaolia et al., 2017). However, the areal requirements per person equivalent for these methods are higher (33-133 times) than CAS and is therefore not applicable in areas with little land capacity (García et al., 2020).

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2.6.1.4 Conventional Disinfection

Disinfection of wastewater has initially been implemented to decrease the high microbial load found in conventional treatment effluent (EPA Victoria, 2002). Traditional disinfection, such as chlorination (Cl) and ultraviolet (UV) radiation, in WWTPs has been seen to exert a variable degree of success in reduction of ARB and ARGs by reducing of the microbial load (Manaia, Macedo, Fatta-Kassinos, & Nunes, 2016; Pang et al., 2016). Cl act as a stressor on the microbiome, leading to cell death and the reduction in the abundance of the ARBs (Lood et al., 2017). UV irradiation impacts the nucleic acids (DNA and RNA) of the bacteria (Rizzo et al., 2013). The irradiation absorbed by bacteria have shown to cause mutations and structural decomposition of the nucleic acids.

Some studies have demonstrated that conventional disinfection not to have any effect on ARG removal (Auerbach, Seyfried, & McMahon, 2007; Munir et al., 2011). However, a study by Zhang et al., (2015) on ARGs sul1, tetX, tetG and intl1 showed a LRV of 1.20-1.49 for Cl and 0.36-0.58 for UV. Variations in efficiency of ARB and ARG removal have been attributed to variable operating conditions (Manaia et al., 2018; Mohammadali & Davies, 2017). Also, ARB resistance to Cl has been documented (Jassim et al., 2015). Cl resistance has led to higher abundance of ARB in the effluent environmental microbiome (Mao et al., 2015). The dosage required for Cl to be effective (>80 mg Cl2min/L) to reduce ARGs have been shown to be higher than the dose generally applied in WWTPs and have also shown to create additional harmful by-products (Zhuang et al., 2015). UV irradiation has been suggested as preferred disinfection method over chlorine (Sharma et al., 2016), due to the damage of plasmids DNA by irradiation, thereby reducing ARG transfer through HGT (Guo et al., 2015). However, UV irradiation for ARG removal is costly due to high irradiation intensities (200-400 mJ/cm²) required for effective ARG removal (Ghernaout, 2020; McKinney & Pruden, 2012).

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2.6.2 Advanced treatment methods

As the demand for water reuse is increasing advanced treatment processes, have been implemented to reduce further the microbial load and removal of emerging contaminants in wastewater effluent in which conventional treatment is insufficient (Ghernaout, 2020). Higher removal rates have been detected in advanced treatment plants, including advanced oxidation processes (AOPS) and advanced membrane technologies (World Health Organization, 2012).

The coupling of membrane processes and AOPs have been proposed by current research (Ghernaout, 2020).

2.6.2.1 Membrane bioreactors (MBR)

MBR is a combination of membrane filtration technology with conventional treatment using bioreactors (Tchobanoglous et al., 2003, p. 913). The degradation of organic pollutants by microorganisms is combined with physical separation provided by the membrane, which results in higher quality effluents (Karaolia et al., 2017, p. 181). This alternative biological treatment method using additional membranes for wastewater treatment has yielded promising reduction values of tested ARGs (Cheng & Hong, 2017; Du et al., 2015; Munir et al., 2011; Ng et al., 2019).

ARG removal in MBRs is mostly related to the physical separation of ARB containing these genes as they are retained by the membrane. A study by Riquelme Breazeal et al., (2013) showed that the permeate (effluent) ARG concentration varied depending on membrane pore size, made evident by enhanced removal by ultrafiltration (UF) compared to microfiltration (MF) in the MBR. The authors further expressed that colloids occur in wastewater matrices and have been seen to enhance ARG removal in membrane treatment. Many bacteria of clinical concern have a larger sized surface than the membrane pores of MF membranes and have thus been removed by size exclusion (Francy et al., 2012; Hai, Riley, Shawkat, Magram, &

Yamamoto, 2014). However, a metagenomic study of MBR effluent in Singapore relating ARG to ARB taxa showed effective removal of ARG, antibiotics and most indicator organisms, but had viable Pseudomonas aeruginosa present with its conferring ARGs (Ng et al., 2019).

Removal of antibiotic resistant genes in wastewater treatment plants

MASTER’S THESIS

REMOVAL OF ANTIBIOTIC RESISTANT GENES IN WASTEWATER TREATMENT PLANTS

MASTER’S THESIS

WATER SCIENCE AND TECHNOLOGY

ENVIRONMENTAL TECHNOLOGY STUDY PROGRAM DEPARTMENT OF MATHEMATICS AND NATURAL SCIENCES

UNIVERSITY OF STAVANGER

2020

ABSTRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

ABBREVIATIONS

1.0 INTRODUCTION

1.1 Objectives

2.0 LITERATURE REVIEW

2.1 Antibiotics

2.2 Antibiotics mode of action

2.2.1 Inhibition of cell wall synthesis

2.2.2 Nucleic acid synthesis inhibition

2.2.3 Protein synthesis inhibition

2.2.4 Folic acid synthesis inhibition

2.2.5 Cell membrane inhibition

2.3 Antibiotic resistance in bacteria

2.3.1 Horizontal gene transfer

2.3.2 Mechanisms of resistance

2.4 Wastewater treatment

2.4.1 Antibiotics in WWTPs

2.4.2 Antibiotic resistance in Wastewater treatment plants (WWTPs)

2.5 Tools for assessing antibiotic resistance in WWTPs

2.5.1 Culture-based methods

2.5.1.1 Agar dilution

2.5.1.2 Broth dilution method

2.5.1.3 Disc diffusion method

2.5.1.4 Etest

2.5.2 Culture-independent methods

2.5.2.1 Polymerase Chain Reaction (PCR)

2.5.2.2 quantitative PCR (qPCR)

2.5.2.3 Viability methods

2.5.3 Metagenomics

2.6 Removal of antibiotic resistance determinants in WWTPs

2.6.1 Conventional treatment

2.6.1.1 Activated sludge

2.6.1.2 Sludge treatment

1.6.1.3 Constructed wetlands

2.6.1.4 Conventional Disinfection

2.6.2 Advanced treatment methods

2.6.2.1 Membrane bioreactors (MBR)